Bacteria employ diverse mechanisms to colonize and co-exist with their eukaryotic hosts. The basis of interaction for many symbiotic and pathogenic bacteria is translocation of multiple effectors into the eukaryotic cellular environment via a type three secretion system (TTSS). Conservation of the TTSS between animal and plant associated microbes highlights the importance of this structure for delivery of effectors across kingdoms. However, it is diversification and evolution of suites of effectors within strains that determines the nature and specificity of interactions with a host. Recent efforts have shown that single strains of bacteria can contain up to 30 effectors, and that the composition of these virulence factors can differ dramatically between isolates of the same species. Despite the importance of effectors in establishing successful infection for human pathogens Salmonella and Yersinia as well as the plant pathogen Pseudomonas syringae, the evolutionary forces that shape the composition of suites of effectors within strains are not well understood. Do effectors generally act independently of one another to cause virulence, or is pathogenesis the outcome of complex interactions between groups of effectors within a host? The goal of this proposal is to understand if and how pairs of effectors interact to promote infection in Pseudomonas syringae. Evolutionary analyses will be used to identify effectors that co-associate with one another between different pathovars of P. syringae. Furthermore, conserved and variable portions of the effector repertoire within one pathovar will be identified through extensive sampling of genetically and geographically diverse isolates using oligonucleotide micro-arrays. Correlation in the presence of effectors through evolutionary time within and between pathovars implies that these effectors interact within the host to cause infection. Once potentially interacting pairs of effectors have been identified, strains containing single and double mutants will be created in order to functionally test for effects on virulence during infection of a host. Relevance To Public Health: Development of disease during infection by many bacteria, such as the human pathogens Salmonella and Yersinia, is entirely dependent on a conserved mechanism employing the translocation of effector proteins from bacterium to host. The goals of this research program are to use evolutionary analyses with the plant pathogen Pseudomonas syringae to identify pairs of effectors that potentially interact within the host and to functionally verify these interactions during infection. This research will therefore provide a greater understanding of the evolutionary forces promoting diversity in suites of effectors within and between bacterial species, and thus virulence within both human and plant hosts.